This invention was made in part with Government funding, and the Government therefore has certain rights in the invention.
This application relates to plant carbohydrate metabolism; in particular, to enzymes which transduce sugar-sensing signals, their encoding genes, and uses thereof.
Sugars are said to be regulatory molecules that are capable of controlling physiology, metabolism, cell cycle, development, and gene expression. Throughout the higher plant life cycle, from germination to flowering to senescence, sugars affect growth and development. Recently, it has become apparent that sugars are physiological signals capable of repressing or activating plant genes that are involved in many essential processes, including photosynthesis, the glyoxylate cycle, respiration, starch and sucrose synthesis and degradation, nitrogen metabolism and storage, pathogen defense, the wounding response, cell cycle progression, pigmentation, and senescence (Sheen, Photosynthesis Res. 39, 427 (1994); Thomas and Rodriguez, Plant Physiol. 106, 1235 (1994); Knight and Gray, Mol. Gen. Genet. 242, 586 (1994); Lam et al., Plant Physiol. 106, 1347 (1994); Chen et al., Plant J. 6, 625 (1994); Reynolds and Smith, Plant Mol. Biol. 29, 885 (1995); Herbers et al., Plant Mol. Biol. 29, 1027 (1995); Mita et al., Plant Physiol. 107, 895 (1995)). Studies in a variety of plant species have also shown that sugar homeostasis appears to be tightly regulated. Elevated sugar concentration leads to stunted growth, reduced photosynthesis, leaf curling, chlorosis, necrotic leaves, and anthocyanin accumulation (Casper et al., Plant Physiol. 79, 11 (1985); von Schaewen et al., EMBO J. 9, 3033 (1990); Dickinson et al., Plant Physiol. 95, 420 (1991); Tsukaya et al., Plant Physiol. 97, 1414 (1991); Sonnewald et al., Plant J. 1, 95 (1991); Huberet and Hanson, Plant Physiol. 99, 1449 (1992); Sonnewald et al., Plant Responses to Sugar Accumulation in Transgenic Tobacco Plants, pp. 246-257, In: M. A. Madore, W. J. Lucas (eds.), Carbon Partitioning and Source-Sink Interactions in Plants, American Society of Plant Physiologists, Rockville, Md., (1995)). In addition, environmental factors such as elevated CO2 and intrinsic genetic variations such as different invertase levels have been proposed to affect photosynthetic capacity through sugar regulation (Stitt, Plant Cell Environ. 14, 741 (1991); Stitt et al., Planta 183, 40 (1991); VanOosten et al., Plant Cell Environ. 17, 913 (1994); Nie et al., Plant Physiol. 108, 975 (1995); Goldschmidt and Huber, Plant Physiol. 99, 1443 (1992)).
By manipulating the expression of a plant hexokinase protein (HXK), we have discovered that this protein is a sensor that mediates diverse sugar responses in plants. In particular, we have engineered transgenic plants that either: (a) express a decreased level of hexokinase protein due to expression of an antisense hexokinase gene and therefore exhibit a decreased sensitivity to sugar; or (b) express an increased level of hexokinase protein and therefore exhibit an increased sensitivity to sugar. Our discovery has broad implications for the manipulation of agricultural crops, for increasing crop yield and quality, and for reducing production costs.
In general, the invention features a method for reducing the level of a plant hexokinase protein in a transgenic plant cell, the method involving expressing in the transgenic plant cell (for example, a cell from a monocot, a dicot, or a gymnosperm) an antisense hexokinase nucleic acid sequence. This produces transgenic plants that are less sensitive to sugar (for example, glucose, sucrose, fructose, or mannose).
In preferred embodiments, the antisense hexokinase nucleic acid sequence is encoded by a transgene integrated into the genome of the transgenic plant cell; the antisense hexokinase nucleic acid sequence includes a plant antisense hexokinase DNA sequence (for example, a sequence that is based on the AtHXK1 nucleotide sequence of FIG. 1F (SEQ ID NO: 3) or the AtHXK2 nucleotide sequence of FIG. 1G (SEQ ID NO: 4)); and the method further includes growing a transgenic plant from the transgenic plant cell, whereby the level of the hexokinase protein is reduced in the transgenic plant.
In related aspects, the invention features a plant cell (for example, a plant cell from a monocot, dicot, or gymnosperm) expressing an antisense hexokinase nucleic acid sequence; and a plant expression vector including an antisense hexokinase nucleic acid sequence, wherein the sequence is operably linked to an expression control region.
In yet another aspect, the invention features a method for increasing the level of a hexokinase protein in a transgenic plant cell, involving expressing in the transgenic plant cell a hexokinase nucleic acid sequence. In preferred embodiments, the hexokinase nucleic acid sequence is from a plant (for example, a DNA sequence that is identical to the AtHXK1 nucleotide sequence of FIG. 1F (SEQ ID NO: 3) or that is substantially identical to the AtHXK2 nucleic acid sequence of FIG. 1G (SEQ ID NO: 4)). This method produces transgenic plants having an increased sensitivity to sugar.
In related aspects, the invention features a substantially pure plant HXK polypeptide including an amino acid sequence substantially identical to the amino acid sequence of AtHXK1 (SEQ ID NO: 1) or AtHXK2 (SEQ ID NO: 2). In preferred embodiments of both of these aspects, the HXK polypeptide is obtained from a plant including, but not limited to, a monocot (for example, rice, corn, wheat, or barley), a dicot (for example, a member of the Solanaceae (for example, potatoes) or a member of the Cruciferae (for example, Arabidopsis, broccoli, cabbage, brussel sprouts, rapeseed, kale, Chinese kale, cauliflower, or horseradish)), and a gymnosperm.
In yet other related aspects, the invention features a substantially pure DNA encoding a plant HXK polypeptide that includes an amino acid sequence substantially identical to the amino acid sequence of AtHXK1 (SEQ ID NO: 1) or AtHXK2 (SEQ ID NO: 2). In preferred embodiments, the DNA includes the nucleotide sequence shown in FIG. 1F (SEQ ID NO: 3) or includes a nucleotide sequence that is substantially identical to the sequence that is shown in FIG. 1G (SEQ ID NO: 4). Such DNAs are obtained from any plant including, but not limited to, a monocot (for example, rice, corn, wheat, and barley), a dicot (for example, a member of the Solanaceae (for example, potatoes) or a member of the Cruciferae (for example, Arabidopsis, broccoli, cabbage, brussel sprouts, rapeseed, kale, Chinese kale, cauliflower, or horseradish)), and a gymnosperm. In other preferred embodiments, the DNAs of the invention are operably linked to a constitutive or regulated promoter.
In yet other related aspects, the invention features a vector including any of the substantially pure DNAs of the invention, the vector being capable of directing expression of the protein encoded by the DNA in a vector-containing cell; a cell, for example, a prokaryotic cell (for example, an E. coli cell) or a eukaryotic cell (for example, a plant cell) which includes any of the DNAs of the invention; and a transgenic plant (or a cell or a seed derived from such a transgenic plant) including any of the DNAs of the invention integrated into the genome of the plant, wherein the DNA is expressed in the transgenic plant.
In various preferred embodiments, the plant cell contains the DNA in the sense orientation and has an increased sensitivity to sugar; the plant cell contains the DNA in the antisense orientation and is less sensitive to sugar; and the DNA is expressed under the control of a constitutive promoter or regulated promoters.
In two other aspects, the invention features a method of producing a plant HXK polypeptide involving: (a) providing a cell transformed with a gene encoding a polypeptide including either an amino acid sequence substantially identical to the amino acid sequence of AtHXK1 (SEQ ID NO: 1) or an amino acid sequence substantially identical to the amino acid sequence of AtHXK2 (SEQ ID NO: 2) positioned for expression in the cell; (b) expressing the plant HXK polypeptide; and (c) recovering the plant HXK polypeptide.
By xe2x80x9chexokinasexe2x80x9d or xe2x80x9cHXKxe2x80x9d is meant a polypeptide that is capable of catalyzing the ATP-dependent conversion of hexoses to hexose-6-phosphates. Methods for assaying such enzymatic activities are known in the art, e.g., those described herein by Renz and Stitt (Planta 190, 166 (1993)).
By xe2x80x9creducing the level of a plant hexokinase proteinxe2x80x9d is meant a decrease in the level of that plant hexokinase protein by at least 30-50%, preferably by 50-80%, and more preferably by 80-95% relative to the level in a control plant (for example, a wild-type plant). Reduction of hexokinase protein levels may be accomplished through the expression of an antisense plant hexokinase nucleotide sequence in a transgenic plant. Levels of plant hexokinase protein are monitored according to any standard technique including, but not limited to, immunoblotting (for example, as described herein). Alternatively, the level of a plant hexokinase protein may be quantified according to standard hexose phosphorylation assays (for example, those described herein).
By xe2x80x9cincreasing the level of a plant hexokinase proteinxe2x80x9d is meant increasing the level of that plant hexokinase protein by at least 50%, preferably 100%, and more preferably greater than 200% relative to the level in a control plant (for example, a wild-type plant). Levels of plant hexokinase protein are monitored according to any standard technique including, but not limited to, immunoblotting (for example, as described herein). Alternatively, the level of a plant hexokinase protein may be quantified according to standard hexose phosphorylation assays (for example, those described herein).
By xe2x80x9can antisense hexokinase sequencexe2x80x9d is meant a nucleotide sequence that is complementary to a plant hexokinase messenger RNA. In general, such an antisense sequence will usually be at least 15 nucleotides, preferably about 15-200 nucleotides, and more preferably 200-2,000 nucleotides in length. The antisense sequence may be complementary to all or a portion of the plant hexokinase mRNA nucleotide sequence (for example, the AtHXK1 and AtHXK2 antisense constructs described herein), and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence binds as well as the length of the antisense sequence will vary, depending upon the degree of inhibition desired and the uniqueness of the antisense sequence. A transcriptional construct expressing a plant hexokinase antisense nucleotide sequence includes, in the direction of transcription, a promoter, the sequence coding for the antisense RNA on the sense strand, and a transcriptional termination region. Antisense HXK sequences may be constructed and expressed as described herein or as described, for example, in van der Krol et al., Gene 72, 45 (1988); Rodermel et al., Cell 55, 673 (1988); Mol et al., FEBS Lett. 268, 427 (1990); Weigel and Nilsson, Nature 377, 495 (1995); Cheung et al., Cell 82, 383 (1995); and U.S. Pat. No. 5,107,065.
By xe2x80x9cless sensitive to sugarxe2x80x9d is meant that the developmental, physiological, or molecular processes that are typically regulated or controlled by internal or external sugar concentrations exhibit reduced responses to the presence of a sugar (for example, glucose, fructose, mannose, or sucrose). For example, a plant having reduced sensitivity to sugar is capable of activating an assortment of genes (for example, photosynthetic genes) that are normally repressed by the presence of sugar, or such a plant is capable of proceeding through its normal developmental pathway even in the presence of sugar concentrations that would otherwise thwart or prevent such development. Analysis of a plant""s sensitivity to sugar is accomplished using a wide variety of bioassays (for example, those described herein). These assays include, but are not limited to, evaluating and monitoring gene expression, seed germination, cotyledon development (for example, cotyledon extension), cotyledon greening, leaf development, embryonic root development, hypocotyl elongation, anthocyanin accumulation, starch accumulation, and time needed for flowering. By comparing phenotypes of wild-type plants and candidate plants (for example, a plant expressing an antisense hexokinase gene), one is readily able to determine whether such a candidate transgenic plant has a reduced sensitivity to a sugar. For example, sugars have been found to repress the expression of both photosynthetic (for example, ribulose bisphosphate carboxylase small subunit and light-harvesting chlorophyll a/b binding protein) and non-photosynthetic (for example, xcex1-amylase, sucrose synthase, malate synthase, and asparagine synthase) genes. Thus, in plants that are less sensitive to sugar, the aforementioned sugar-repressible genes have a decreased, reduced, or attenuated level of sugar-mediated repression.
By xe2x80x9cincreased sensitivityxe2x80x9d is meant that the developmental, physiological, or molecular processes that are typically regulated or controlled by internal or external sugar concentrations exhibit increased or elevated responses to the presence of a sugar (for example, glucose, fructose, mannose, or sucrose). For example, a plant having increased sensitivity to sugar is capable of elevating, raising, or promoting the activation of an assortment of genes (for example, vegetative storage proteins) that are normally activated by the presence of sugar. Analysis of a plant""s sensitivity to sugar is accomplished using a wide variety of bioassays. These assays include, but are not limited to, evaluating and monitoring gene expression, seed germination, cotyledon development (for example, cotyledon extension), cotyledon greening, leaf development, embryonic root development, hypocotyl elongation, anthocyanin accumulation, starch accumulation, and time needed for flowering. By comparing phenotypes of wild-type plants and candidate plants (for example, a plant expressing at least one additional copy of hexokinase gene), one is readily able to determine whether such a candidate transgenic plant has an increased sensitivity to a sugar. For example, sugars have been found to activate the expression of genes such as nitrate reductase, xcex2-amylase, sucrose synthase, and potato storage protein. Thus, in plants exhibiting an increased sensitivity to sugar, the aforementioned sugar-inducible genes have an increased, elevated, or heightened level of sugar-mediated expression.
By xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cproteinxe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By xe2x80x9csubstantially identical to AtHXK1xe2x80x9d is meant a plant hexokinase polypeptide that includes an N-terminus which is at least 50%, preferably 75%, more preferably 85-90%, and most preferably 95% identical to the N-terminus of AtHXK1 (amino acids 1-61 of FIG. 1B; SEQ ID NO:1). The length of comparison will generally be at least 15 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 60 amino acids.
By xe2x80x9csubstantially identical to AtHXK2xe2x80x9d is meant a plant hexokinase polypeptide or nucleic acid sequence that exhibits at least 86%, preferably 90%, more preferably 95%, and most preferably 99% identity to the amino acid or nucleic acid sequences of AtHXK2 (FIGS. 1B and 1G; SEQ ID NOS: 2 and 4).
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), BLAST, or PILEUP/PRETTYBOX programs). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a plant hexokinase polypeptide (for example, AtHXK1 or AtHXK2) which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, plant hexokinase polypeptide. A substantially pure plant hexokinase polypeptide may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding a plant hexokinase polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) an HXK polypeptide (for example, AtHXK1 or AtHXK2).
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a plant hexokinase polypeptide such as AtHXK1 or AtHXK2, a recombinant protein, or a RNA molecule).
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, or hormone-inducible elements); such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cplant cellxe2x80x9d is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genomes.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.